This invention concerns transgenic plant cells and plants, a method for their preparation and the use of invertase inhibitor cDNA sequences in an antisense or sense orientation to produce such plants.
Improvement in the quality and quantity of plant reserve material in seeds of dicotyledonous and monocotyledonous agriculturally-useful plants represents an important objective of biotechnology research. Hitherto, strategies generally were developed which were based on the introduction of particular genes whose genetic products constituted enzymes which are themselves involved in the synthesis of the energy reserves (e.g. ADP glucosepyrophosphorylase). Furthermore, methods are also described, in which an increased rate of glycolysis is obtained by modified expression of heterologous, and therefore, deregulated invertases or glucokinases in the cytosol (DE-A1-195 29 696). In later variants the increased breakdown of sucrose by a deregulated fungal invertase, in combination with a deregulated bacterial glucokinase, leads to an increased rate of glycolysis. This approach rests on the assumption that the synthesis of stored oils in seeds is stimulated because of the increased concentrations of the intermediates of glycolysis, since the metabolisation of the primary photoassimilate, sucrose, is required for phosphorylised hexoses or the fatty acid precursors, pyruvate and acetyl coenzyme A.
DE-A1-195 29 696 correspondingly describes the introduction of a foreign, e.g. fungal gene for the expression of the invertase. Because of the supply of the foreign gene, this fungal invertase enzyme (which is foreign and therefore is not subjected to any regulation) is formed in an amplified manner by regulating a suitable promoter, by means of which the decomposition of the sucrose catalysed by the invertase into glucose and fructose occurs faster. The resulting production of glucose at a higher rate is to bring about in the end an accelerated production of plant reserve material. This process is based on an intervention in the metabolism in the cell of the seed storage tissue, whereby the assimilate transfer between maternal and seed parenchyma is affected only indirectly.
The importance of cell wall invertase for the development of seeds high in starch and protein is well-known. Thus for example the starch accumulation in corn seed is adversely affected with reduced expression of a cell wall invertase by interference in the assimilate transfer between pedicel and endosperm. Flower-specific cell wall invertase isoforms for different plant species are well-known. For Nicotiana tabacum, it was able to be shown that an apoplastic invertase inhibitor is powerfully expressed, particularly in the ovary and stamens. Greiner et al. (Plant Physiol. (1998), 733-742) disclosed the amino acid sequence and cDNA sequence of the mentioned invertase inhibitor as well as its in vitro demonstration of function by means of a heterologous expressed inhibitor protein. However, in vivo inhibition was still not indicated. Moreover, it is well-known that varying isoforms of cell wall invertases and invertase inhibitors exist in different tissues and at different times in plant development.
A specific classification of the activities and their possible combined effects of these two time-specific and tissue-specific occurring proteins has not been feasible until now. There are no known studies of an in vivo situation with regard to the regulation of cell wall invertases by invertase inhibitors. Just as little known, was if and when which isoforms of the cell wall invertases are subjected to endogenous regulation by invertase inhibitors during the seed development and if so, which isoforms of the invertase inhibitors. That is why the specific use of these proteins for the production of beneficial plants has not been possible hitherto.
The technical problem of the invention therefore is to provide transgenic plant cells, plants and a method of producing these, in which the plants are characterised by the production of seeds which, compared to seeds of untransformed plants, have a greater amount of plant reserve material such as carbohydrates, fats or proteins, without endogenous or exogenous proteins being over-expressed and without the phenotype of the plant and its development being impaired.
The technical problem underlying this invention is provided by a process for producing a transgenic plant with a deregulated invertase activity which stimulates plant development, whereby the process provides for: a nucleotide sequence of an invertase inhibitor to be produced from a cDNA bank of a cell suspension culture or from flowers with young ovules from a plant, or to be derived therefrom; a plant cell of a plant of the same type or variety with a DNA construct, containing the functional nucleotide sequence of an invertase inhibitor bound to at least one regulatory unit to be transformed, cultivated and regenerated to a plant whose seed produces a greater amount of reserve material such as carbohydrates, fat or protein in comparison with plants not transformed with such a DNA construct.
The invention provides in particular for the production of transgenic plants with a modified expression of an invertase inhibitor, preferably an apoplastic invertase inhibitor, whereby the plants are characterised by the expression of invertase inhibitor proteins being reduced or completely eliminated during seed development. The process can be applied advantageously to the most widely different dicotyledonous or monocotyledonous useful plants, for example: rape, sunflower, peanut, oil palm, soy bean, Calendula officinalis, Coriandrum sativum, Crambe abyssinica, Cuphea ssp., Dimorphotheca pluvialis, Euphorbia lagascae, Euphorbia lathyris, Lesquerella grandiflora, Limnanthes alba, Linum usitatissimum, Lunaia annua, Lunara biennis, Oenothera ssp., Ricinus communis, and Simmondsia chinensis as plants with seeds storing fat; corn, rice, wheat, barley, oats, and rye as plants with seeds storing starch; and soy bean or pea, for instance, as plants with seeds storing protein.